A viability assay showed that DA could inhibit tumor cell growth in a time- and dose-dependent manner. Flow cytometry showed that DA blocks the cell cycle at G0/G1 phase. Western blotting results demonstrated that the induction of apoptosis by DA correlated with the induction of pro-apoptotic proteins Bax, and Bid, and a decrease in the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl. Furthermore, treatment of HCT-116 bearing nude mice with DA significantly retarded the growth of xenografts. Consistent with the results in vitro, the DA-mediated suppression of HCT-116 xenografts correlated with Bax and Bcl-2. Taken together, these results suggest that DA could be a novel and promising approach to the treatment of CRC.In traditional Chinese medicine, shikonin and its derivatives, has been used in East Asia for several years for the prevention and treatment of several diseases, including cancer. We previously identified that β,β-dimethylacrylshikonin (DA) could inhibit hepatocellular carcinoma growth. In the present study, we investigated the inhibitory effects of DA on human colorectal cancer (CRC) cell line HCT-116  Although advances have been made in clinical medicine, no effective treatment modalities are available for patients with advanced or metastatic tumors . ColorecLithospermum erythrorhizon that belong to the Boraginaceae family  is isolated mainly from the roots of e family  4]. The. TheLithIn the present study, we investigated the efficacy of DA in CRC cells and nude mice tumor models with the aim of developing a novel CRC treatment approach.HCT-116 cells were treated with DA and the cell viability was determined by MTT assay. The treatment of HCT-116 cells for 24–72 h with 5, 10 and 15 μg/mL of DA resulted in cell growth inhibition in a dose- and time-dependent manner . To conf0/G1 phase of the cell cycle in a dose-dependent manner.To investigate the effect of DA on cell cycle distribution of HCT-116 cells, the DNA content was analyzed by flow cytometry. As shown in HCT-116 cells were treated with 5, 10 and 15 μg/mL of DA for 48 h. After treatment, the degree of apoptosis was measured. The induction of apoptosis was found to be dose-dependent . These rThe Bcl-2 family plays a major role in the regulation of apoptosis by functioning as promoters or inhibitors of cell death. We therefore examined the expression of Bcl-2 family proteins in DA-treated HCT-116 cells in a dose-dependent manner. As shown in To determine whether systemic therapy with DA could stunt tumor growth in animals, we established HCT-116 xenografts in nude mice. As shown in DA was obtained from Huakang Pharmaceutical Company  and the purity was determined by high-performance liquid chromatography.2 at 37 °C and were sub cultured at an initial density of 1 × 105 cells/mL every three days. All experiments were performed with cells in the logarithmic phase of growth.Human CRC cell lines HCT-116 were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The cells were cultured in RPMI-1640 media  supplemented with 10% fetal bovine serum  and antibiotics . All cells were grown in 5% CO3) were seeded in a 96-well plate and subsequently treated with DA  for 12, 24 and 48 h. Twenty micro liters of MTT (5 mg/mL) solution  were added to each well at 37 °C for four hours. After removing the media, 150 μL of dimethylsulfoxide (DMSO) were added and left for 20 min at room temperature to dissolve the formazan crystals in a shaker. The absorbance at 570 nm was measured in a micro plate reader .The cells  and 0.1 mg/mL RNase . After 30 min at 37 °C, the cells were analyzed with a flow-cytometer and the cell cycle was determined. Experiments were performed in triplicate and repeated three times.To estimate the proportion of cells in the different phases of the cell cycle, the cell DNA contents were measured using flow cytometry. HCT-116 cells plated on 20-cmThe cell death detection ELISA kit  was used for assessing apoptosis according to the manufacturer’s instructions. Briefly, cells were treated with DA for 48 h. After treatment, the cells were lysed and the cell lysates were overlaid and incubated in a micro titer plate modules coated with anti-histone antibody for detection of apoptosis.Western blotting was performed as we have previously described . Briefly7 cells/mL, 0.2 mL/mouse) were injected subcutaneously into the upper left flank region of mice. The mice were randomly divided into four groups (n = 6 per group). Mice in each group were treated daily with DA  or the same volume of saline as a negative control group by intraperitoneal administration every alternate day from day one. The dose of DA was based on our previous work that did not show any cytotoxic effect in normal mice. All the mice were sacrificed on day 13 after inoculation with HCT-116 cells. The tumor was measured for largest (a) and smallest (b) diameters, and the tumor volume was calculated as V = a × b2/2. Tumor inhibitory rates were calculated with the following formula: tumor inhibitory rate (%) = 1 − (tumor weight of treated group/tumor weight of control group) × 100%. Guidelines for the humane treatment of animals were followed as approved by the Sichuan University.Male nude mice (4- to 5-week old) were obtained from the Sichuan University Animal Center. Mice were housed in temperature-controlled rooms with a 12-h alternating light-dark cycle. HCT-116 cells  solution depended on the sample condition. The stained slides were visualized on a microscope. Images were captured with an attached camera linked to a computer.p < 0.05 was considered statistically significant.The results are expressed as the mean ± SD. Multiple comparisons were performed by one-way ANOVA followed by Duncan’s post-hoc test using SPSS 18.0 . A value of Lithospermum erythrorhizon have been proven to have antitumor activity against a number of cancer cells, and DA is one of the most effective agents. Previous studies demonstrated that shikonin and its derivatives exerted inhibition effects on several tumor cell lines, including human premyelocytic leukemia cell line HL-60, human breast cancer cell line MCF-7, human oral squamous cell carcinoma cell line Tca-8113, human lung adenocarcinoma cell line A549 and so on [in vivo animal experiments such as S180 sarcoma model and Lewis lung carcinoma model [in vivo.Many compounds extracted from nd so on . The antma model ,16. Howein vitro. Moreover, DA could suppress the tumor growth of HCT-116 xenografts. Generally, tumorigenesis and tumor progression are strongly associated with apoptosis.In the present study, we found that DA inhibited the proliferation of human CRC cell line in a dose- and time-dependent manner 0/G1 phase. We also found that DA could induce apoptosis, a finding confirmed by Histone/DNA ELISA kit.We firstly analyzed the effect of DA in human colorectal cancer cell line HCT-116. Consistent with our previous studies, DA caused significant inhibition of tumor growth. Flow cytometric analysis of the effects of DA on the cell cycle in treated HCT-116 cells revealed a dose-dependent decrease in cell proliferation and a concomitant accumulation of cells in GWe also investigated the underlying mechanisms for the apoptosis induction of CRC cells. Apoptosis, or programmed cell death, is an essential physiological process that is required for normal development and maintenance of tissue homeostasis . Initiatin vivo. Our results indicated that treatment with DA could inhibit the growth of the HCT-116 xenografts in a dose-dependent manner. Consistent with the results in vitro, the immunohistochemical staining showed that DA decreased the expression of Bcl-2 and increased the expression of Bax.Next, we conducted experiments designed to test the potential for DA to exert protective effects against colorectal cancer in vitro and in vivo by inducing apoptosis involved in down-regulation of Bcl-2 expression and up-regulation of Bax expression. Our study suggests that DA represents a promising novel agent that should be developed for the treatment of CRC.In summary, we presented experimental evidence, which strongly supports the antitumor effects of DA in CRC


Photoluminescence of highly structured ZnO layers comprising well-shaped hexagonal rods is presented. The ZnO rods  were grown in air onto a preheated soda-lime glass (SGL) or ITO/SGL substrate by low-cost chemical spray pyrolysis method using zinc chloride precursor solutions and growth temperatures in the range of 450-550°C. We report the effect of the variation in deposition parameters  on the photoluminescence properties of the spray-deposited ZnO nanorods. A dominant near band edge (NBE) emission is observed at 300 K and at 10 K. High-resolution photoluminescence measurements at 10 K reveal fine structure of the NBE band with the dominant peaks related to the bound exciton transitions. It is found that all studied technological parameters affect the excitonic photoluminescence in ZnO nanorods.PACS: 78.55.Et, 81.15.Rs, 61.46.Km NRL), is a material with large effective surface area, suitable for short-wavelength devices, such as ultraviolet (UV) light-emitting diodes (LED) [NRL can be used as an antireflection coating on silicon solar cells [NRL was used in ETA solar cells showing energy conversion efficiency of 4.2% [ZnO is a semiconductor material for various photonic and electrical applications. ZnO shows a unique set of physical and chemical properties, such as a wide band gap 3.37 eV), large exciton binding energy 60 meV) at room temperature, radiation hardness , piezoel.37 eV, les (LED) , UV nanoes (LED) , UV phot0 meV at es (LED) , and chees (LED) ,10. As aar cells , surfacear cells  or with ar cells . The che of 4.2% .NRL grown via a low-temperature wet-chemical methods  do not show a high excitonic to visible emission intensity ratio at 300 K (room temperature), indicating a high defect concentration in these samples [NRL deposited by the wet-chemical methods can be improved by post-growth annealing at high temperatures of 200-850°C in forming gas or vacuum environment [For many of such devices, a large-scale, low-cost fabrication of high optical and crystalline quality ZnO is desirable. Low-temperature chemical synthesis methods can provide large scale and low-cost fabrication. Photoluminescence (PL) is a very sensitive and an effective method to identify the dominant recombination mechanism and defects in materials. However, according to PL study, the as-deposited ZnO samples -21. The ironment ,17-19,21NRL, grown via CSP at growth temperatures up to 550°C from zinc chloride (ZnCl2) solutions. CSP technique is a template- and catalyst-free method, allowing fast and low-cost deposition of ZnONRL [NRL comprise c-axis (002)-oriented, hexagonal rods of pure ZnO wurtzite phase with aspect ratio up to 30 [NRL. As a result, the PL properties were found to depend on the growth temperature, substrate type, spray rate, and the solvent type. The correlation between the electrical and the photoluminescence properties of the CSP-deposited ZnONRL were reported earlier [NRL were focused on the development of the ZnONRL [NRL was reported until now.In this study, strong excitonic PL is observed in the as-deposited ZnOf ZnONRL . Accordiup to 30 ,24. The  earlier . Our pree ZnONRL -24, no sNRL were deposited by pneumatic CSP method in air at growth temperatures (TG) of 480°C, 530°C, 550°C using zinc chloride (ZnCl2) precursor solutions. Two kind of substrates were used: soda-lime glass (SGL) and commercial indium tin oxide-covered glass (ITO/SGL). The growth temperature TG was controlled through the temperature of a molten tin bath used to obtain uniform heating of the substrate. Other deposition parameters were varied in the following: Three discrete values were used for the spray rate (v): 1.2, 2.2, and 6.2 ml/min. Two kinds of solvents for ZnCl2 were used: H2O and alcoholic solvent , the volume of spray solution was 50 ml. The concentration of ZnCl2 in the spray solution was kept constant in the presented series ranging from 0.05 mol/L ( Effect of the substrate on PL properties of ZnONRL and Effect of the spray rate on PL properties of ZnONRL sections) to 0.1 mol/L . The acidity of the solution was kept at pH = 5. The studied samples were as grown. XRD measurements were performed on a Rigaku Ultima IV diffractometer with Cu Kα radiation (λ = 1.5406 Å) using the silicon strip detector D/teX Ultra.The ZnONRL were made at room temperature (T = 300 K) and at T = 10 K in a closed-cycle He cryostat (Janis). He-Cd laser (325 nm) was used as an excitation source, the excitation intensity was approximately 0.5 mW/mm2. The luminescence emission in the energy region of 1.45-3.45 eV was dispersed by a computer-controlled Carl Zeiss SPM-2 monochromator (f = 0.4 m) equipped with a prism and detected by FEU-79 photomultiplier with a lock-in amplifier. High-resolution LabRam Horiba Yvon HR 800 spectrometer and CCD-detector were used in the region of 3.30-3.40 eV for a closer study of the PL of ZnONRL in UV-region. The same apparatus was used for the Raman spectroscopic studies. Zeiss EVO-MA15 apparatus was used for the scanning electron microscopic (SEM) and the energy dispersive spectroscopic (EDS) study of the ZnONRL. The apparatus was equipped with the Oxford Instruments PentaFet x3 spectrometer using the INCA Energy EDS system at accelerating voltage of 7 kV.The photoluminescence (PL) measurements of ZnONRL is presented in Figure NRL are highly c-axis-oriented hexagonal (wurtzite) ZnO structures. The Raman spectrum of as-deposited ZnONRL is presented in Figure -1 with a fitted excitonic peak widths (FWHM) of 1.2 and 6.1 cm-1, respectively, are dominant ones. Raman peaks at 99, 438, and 379 cm-1 are attributed to the E2(low), E2(high), and A1(TO) Raman modes of wurtzite phase of ZnO, respectively [-1 which is correlated to VO and/or Zni defects [-1 are due to the second order or multiple phonon scattering of the Raman modes of the ZnO wurtzite structure, observed by other authors as well [NRL is ca. 1.5. The excess of oxygen (compared to the stoichiometric ZnO) as well as the presence of In, Sn, and Si signal in the EDS spectra (not presented), originates from the ITO/SGL substrate. Other elements are not detected by the EDS.Typical XRD pattern of sprayed ZnOectively . The Ram defects  is not o as well . The shaNRL grown onto ITO/SGL substrate are presented as Figure NRL deposited from aqueous solution of ZnCl2 (0.1 mol/L) at TG = 550°C is composed of separately standing ZnO crystals with a diameter of 100-300 nm and length of ca. 800 nm, see Figure TG = 480°C using aqueous solutions, the rod-like shape of the crystals is not well-developed  emission at T = 10 K and at T = 300 K, see Figure NRL, Effect of the growth temperature on PL properties of ZnONRL, Effect of the spray rate on PL properties of ZnONRL, and Effect of solvent type on PL properties of ZnONRL sections). The NBE peak positions were determined by fitting the spectra with the Lorentz distribution using the Fityk curve-fitting freeware. The NBE peak measured at T = 300 K is due to free exciton transition. High-resolution PL measurements at T = 10 K reveal four transitions due to bound excitons: a prevailing exciton peak at 3.358-3.360 eV, two other exciton peaks at 3.363 eV and 3.368-3.370 eV, a peak of the two electron transition at 3.334-3.335 eV and the free exciton peak at 3.378 eV [The PL spectra of the best ZnO3.378 eV ,29-32. T3.378 eV ,29-32, iNRL deposited at TG of 550°C onto the SGL and the ITO/SGL substrates, are presented as Figure NRL on ITO/SGL substrate. In addition, the green PL emission band in the region of 2.2-2.8 eV is not present at T = 10 K and at T = 300 K in case of the ITO/SGL substrate used. This could be expected because strong red and green bands do not tend to co-exist [The PL spectra of ZnOco-exist ,33.O [Zn [i, OZn, and VO [i, Oi [i [The broad and structureless green luminescent band is mostly reported as an intrinsic defect level due to VO ,35 or VZO [Zn  or both,O [Zn . Althoug, and VO . The oriO [i, Oi ,18 whilei, Oi [i -40. Withi, Oi [i .T = 300 K), for the ZnONRL grown at 550°C onto ITO/SGL, no green emission is detected, see Figure NRL with a lower content of defects can be deposited using the ITO instead of SGL substrates, however, such a growth mechanism of the ZnONRL onto the ITO remains unclear. It may be speculated that a more homogenious lateral heat distribution may be responsible for the significant improvement of the NBE to red band intensity ratio (INBE/IRED) in the case of using the ITO/SGL substrate for deposition of ZnONR. Another speculation may be that ITO acts as a barrier, restraining the diffusion of some elements, e.g., sodium from the SGL glass during high-temperature spray process as it has been recorded by X-ray photoelectron spectroscopy for the CSP-deposited CuInS2 films [NRL with high crystal quality during the CSP process. This observation is of significant importance due to the fact that for many electronic applications including solar cells, a conductive and transparent material, such as ITO, is required as the substrate material.At room temperature  can be correlated to a very low concentration of defects [NRL results in an increase of the ratio of NBE to red band intensity  could be estimated from Figure NRL deposited at TG = 480°C. At the same time, an increase of the excitonic PL band was registered without a decrease of the FWHM of the fitted peaks . The use of an alcoholic solution results in a smaller spray droplet size, leading to a more homogeneous distribution of the sprayed solution over the substrate, compared to an aqueous solution. In addition to a smaller droplet size, the high volatility of the alcoholic solvent is very likely to cause a lower rate of the substrate cooling. Thus, a higher value of the effective growth temperature is expected in the reaction zone of pyrolysis, resulting in a somewhat higher growth temperature than the stated TG. This can be discussed in the following.In case the alcoholic solvent is used, the excitonic band intensity increases approximately 30 times. The PL intensity ratio INRL deposited at 480°C . The use of an alcoholic instead of aqueous solution could be applied in order to achieve a higher surface-to-volume ratio at a similar TG. It must be pointed out, however, that the PL of the ZnONRL deposited at a low spray rate (1.2 ml/min) from aqueous solution was superior to all considered samples in sense of narrow NBE peaks (Effect of the spray rate on PL properties of ZnONRL section).Although the density of rods and the aspect ratio is known to increase when deposited from alcoholic solutions , the effNRL), grown via fast and low-cost pneumatic CSP method in air. The results indicate that a good crystal quality can be achieved, while ZnONRL with the best optical quality were deposited at a growth temperature of 550°C using ITO/SGL substrate. No green or red emission was detected for these samples, measured at 10 K. In addition, green emission is absent at 300 K for the best samples. The prevalence of the bound exciton transitions suggests a low defect density in our best ZnONRL. Low spray rates (1.2 ml/min) are recommended in case aqueous solution is sprayed, otherwise alcoholic solution is preferred for the deposition of ZnONRL. Lower substrate temperature is acceptable in case alcoholic solution is used, in order to achieve comparable crystal quality, compared to the use of aqueous solution. At T = 10 K, fitted excitonic peak widths (FWHM) are 2-5 meV. For comparison, peak widths of 6-9 meV are reported for ZnONRL grown by hydrothermal method with post-deposition annealing [NRL is superior compared to other solution-based methods in the sense of photoluminescence properties. The results of this study imply that the spray-deposited ZnONRL is sufficient for the use as a transparent window layer in an ETA solar cell and for applications to other low-cost photonic devices at room temperature.We report photoluminescence properties in as-deposited ZnO nanorod layers (ZnOnnealing . These rThe authors declare that they have no competing interests.EK carried out photoluminescence experiments, analysis, and interpretation of experimental results and writing of major part of paper, TR carried out photoluminescence experiments, TD designed and deposited ZnO nanorods, JK provided valuable theoretical discussions, AM provided valuable experimental discussions, VM carried out SEM study, MK designed the study, revised the manuscript, provided motivation and discussions. All authors read and approved the final version of the manuscript.


Reduction of proteinuria and blood pressure (BP) with blockers of the renin-angiotensin system (RAS) impairs the progression of chronic kidney disease (CKD). The aldosterone antagonist spironolactone has an antiproteinuric effect, but its use is limited by side effects. The present study evaluated the short-term antiproteinuric effect and safety of the selective aldosterone antagonist eplerenone in non-diabetic CKD.Open randomized cross-over trial.Forty patients with non-diabetic CKD and urinary albumin excretion greater than 300 mg/24 hours.Eight weeks of once-daily administration of add-on 25–50 mg eplerenone to stable standard antihypertensive treatment including RAS-blockade.24 hour urinary albumin excretion, BP, p-potassium, and creatinine clearance.The mean urinary albumin excretion was 22% , P<0.001, lower during treatment with eplerenone. Mean systolic BP was 4 mmHg , P = 0.002, diastolic BP was 2 mmHg , P = 0.02, creatinine clearance was 5% , P = 0.005, lower during eplerenone treatment. After correction for BP and creatinine clearance differences between the study periods, the mean urinary albumin excretion was 14% , P = 0.008 lower during treatment. Mean p-potassium was 0.1 mEq/L  higher during eplerenone treatment, P<0.001. Eplerenone was thus well tolerated and no patients were withdrawn due to hyperkalaemia.Open label, no wash-out period and a moderate sample size.In non-diabetic CKD patients, the addition of eplerenone to standard antihypertensive treatment including RAS-blockade caused a moderate BP independent fall in albuminuria, a minor fall in creatinine clearance and a 0.1 mEq/L increase in p-potassium.NCT00430924Clinicaltrials.gov  Reductions in blood pressure (BP) and urinary albumin excretion in chronic kidney disease (CKD) have been shown to reduce the risk of cardiovascular (CV) events and improve renal outcome. A reduction in proteinuria is considered a surrogate marker of reduction in CKD progression rate and residual proteinuria has influence on the course of progression to end stage renal disease Inhibition of the renin-angiotensin system (RAS) attenuates the progression of diabetic and non-diabetic CKD in patients with proteinuria and hypertension in excess of the BP lowering effect alone All patients were included after written informed consent. The study was approved by The Ethical Committee of Copenhagen County and the Danish Medicines Agency. The study was carried out according to the Helsinki Declaration.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see The study was carried out in a randomized, open-label, cross-over design comparing an 8-week control period with an 8-week period of once-daily administration of eplerenone. Randomization was done by the principal investigator drawing sealed opaque envelopes. Patients were either allocated to start in the intervention period followed by the control period or vice versa with no wash-out period in between.Inclusion criteria were: age >18 years, persistent 24 hour proteinuria, initially planned as >2000 mg, but after inclusion of the first patient changed to >500 mg or albuminuria >300 mg, BP>130/80 mmHg or ongoing stable antihypertensive treatment, including RAS-blockade. There was no demand for ongoing RAS-blocking therapy.Exclusion criteria were: diabetic nephropathy, creatinine clearance <20 mL/min, plasma (p-) potassium >5.0 mEq/l, allergy to aldosterone antagonists, chronic liver insufficiency, ongoing treatment with CYP3A4-inhibitors, lithium or immunosuppressive agents including steroids, invalidating psychiatric disorders, other severe non-renal disease, woman of childbearing potential not using safe contraception, pregnancy or breast-feeding. Patients were recruited from and followed in the outpatient clinics of the two participating departments. All patients were seen by the principal investigator.Eplerenone treatment was initiated by a once daily oral dose of 25 mg administered as add-on treatment to ongoing therapy. The dose was doubled after one week to 50 mg once daily for seven weeks. Patients were seen at weeks 0, 1, 2, 4, 8, 9, 10, 12 and 16. The BP goal was <130/80 mmHg. In case of symptomatic hypotension, reductions were primarily made in non-RAS-blocking antihypertensive agents and in case of BP above target non-RAS-blocking agents were added. Main outcome variables were albuminuria based on single 24 hour urine samples collected for every visit, fractional excretion of albumin, BP, p-potassium, and creatinine clearance, which were measured at each visit.Potassium supplements were withdrawn before initiation of eplerenone treatment. There were no dietary restrictions at trial entry. Withdrawal criteria were any CV event, serious non-renal disease, a persistent rise in p-creatinine above 30% of baseline value, persistent hyperkalaemia, defined as p-potassium >5.5 mEq/L at two successive visits, or pregnancy despite safe contraception.BP was measured using the mercury sphygmomanometer auscultatory method. Cuff width was selected according to arm circumference. BP measurements were done after 5–10 minutes of rest in the sitting position. Analysis of urinary albumin was done by immunoturbidimetry with antibodies from Dako A/S (Konelab PRIME 60), inter-series coefficient of variation (CV) 2% at Herlev Hospital and Tinaquant from Roche (Roche Modular), inter-series CV 10%, at Rigshospitalet.P-creatinine was analyzed by the enzymatic method, MDRD-IDMS calibrated (Vitros 5.1), inter-series CV 2% at Herlev Hospital and by Roche Modular Enzymatic method, MDRD-IDMS calibrated, inter-series CV 2.1% at Rigshospitalet.Fractional albumin excretion was calculated according to the following formula:A reduction of albuminuria of 25% was considered as the minimal clinically relevant response to eplerenone treatment. Sample size was estimated to be a total of 40 patients, (power 0.8 and level of significance 0.05). Data are presented as mean and standard deviations (SD). Where data were not normally distributed, logarithmic transformations were made and these data are presented as well as geometric means with 95% confidence intervals [CI]. Changes in logarithmic transformed parameters were calculated as ratios and additionally shown in percent.The treatment effects were calculated by the paired samples t-test, using the mean value of the variable over all four visits in each period – i.e. control and treatment. Analyses of values at individual visits were done as a secondary end-point. Before analyzing treatment effect, analyses of carry-over and time effect were made. To test for carry-over effect, we conducted a two-sample t-test between the two randomization groups using the mean value of each subject. For time effect a paired samples t-test was conducted comparing values for each patient at the two different time periods. Two-way ANOVA was used for testing the treatment effect on urinary albumin excretion when adjusting for creatinine clearance and systolic BP. One-way ANOVA was used for testing differences in response to treatment, when level of RAS-blockade or baseline creatinine clearance was taken into account. Evaluation of significance over multiple periods was done by repeated measure ANOVA. Comparisons of data from individual visits were corrected for multiple comparisons by the Bonferroni method. Since only baseline data were tested against data from each of the four following visits, correction was made by multiplying the P-values by four. When applying correction for multiple testing the corrected P-values are shown. All P-values were two-sided and P-values<0.05 were regarded as significant. All confidence intervals are two-sided 95% CI. The statistical analyses were done using the SPSS statistical software, version 17.0 and R statistical software version 2.12.1, 2011, R Foundation for Statistical Software, Vienna, Austria.Forty-two patients were included in the study. One was included by mistake fulfilling the exclusion criterion of serious non-renal disease and was withdrawn after the first visit. Another patient was withdrawn due to non-compliance with the urinary collections. Thus, the statistical analysis comprises forty patients. Two patients were withdrawn before completion, one due to myocardial infarction  and one due to insomnia . Data from these two participants were averaged for the visits they had completed. Thirty-eight patients completed the full study protocol; one patient did not tolerate 50 mg eplerenone, as described below in the safety results. All patients demonstrated very good compliance, assessed by tablet counts. The trial was initiated in April 2007 and completed and ended as planned in August 2009.Renal diagnoses were based on previous renal biopsies in chronic glomerulonephritis (n = 26), specified in Creatinine clearance ranged from 24 to 195 mL/min at the time of inclusion and patients were classified as CKD stages 1 (n = 18), 2 (n = 10), 3 (n = 10) and 4 (n = 2). All patients but two were on RAS-blockade. One patient received vitamin D supplements. Baseline patient characteristics of all patients are presented in Albuminuria was significantly lower during the add-on eplerenone period as compared with the control period with a 22% , P<0.001, lower excretion, as shown in Fractional albumin excretion was significantly lower in the eplerenone period, see When systolic BP reduction was taken into account, the urinary albumin excretion was 17%  lower during eplerenone treatment, P<0.001. When corrected for the lower creatinine clearance during the treatment period, the reduction in albumin excretion was 19% , P = 0.0003. When both BP and creatinine clearance were included in the statistical model, the total difference in 24 hour urinary albumin excretion was 14% , P = 0.008.As there was no wash-out period between study periods, additional analysis was done excluding the first measurements in each block (i.e. visit 2 and 6). Excluding these measurements the difference in urinary albumin excretion was 25% , P<0.001. Comparing only visits 2 and 6, there was no difference in albumin excretion between treatment and control periods, as well as there was no detection of any significant carry-over effect, P = 0.3.To study the influence of baseline RAS-blockade on the antialbuminuric effect of eplerenone baseline RAS-blockade was separated into three levels for post hoc analysis: Low level blockade when the daily dose of ACEi/ARB was <¼ of the maximum dose for the individual drug (n = 11), moderate level blockade when the ACEi/ARB dose was between ¼ and ½ of the maximum dose (n = 18) and high level blockade when the ACEi/ARB dose was >½ the maximum dose (n = 2) or in case of combined ACEi and ARB (n = 7). There was a non-significant tendency for patients on moderate RAS-blockade to have a greater response on eplerenone than the other groups. Patients on combined ACEi and ARB had no significant reduction in albuminuria during eplerenone treatment.Systolic and diastolic BP was significantly lower during add-on eplerenone treatment when compared to the control period . There wThere were no significant differences between diastolic BP at the end of the two periods. There were no significant differences in BP reduction between groups of low, moderate or high baseline RAS-blockade.There were no significant carry-over, P = 0.4 and P = 0.9, or time effects, P = 0.5 and P = 0.2 for systolic or diastolic BP.As shown in Four patients reported increased dizziness during the treatment period of the study compared to their usual state without any change in BP. None required reduction in dose of eplerenone due to dizziness alone.No patients were withdrawn due to hyperkalaemia or other adverse events. The mean difference in p-potassium was 0.1 mEq/L, P<0.001 between the eplerenone and control periods, as shown in Four percent of all p-potassium values measured were above 5.0 mEq/L and none were above 6.0 mEq/L. Only one patient had a single p-potassium value above 5.6 mEq/L at a planned visit. Consequently the patient was seen at an extra control, where p-potassium had increased to 5.8 mEq/L. Because of this, as well as symptomatic hypotension, nausea and general discomfort the dose of eplerenone was reduced to 25 mg daily. The patient completed the study without further adverse effects.There were no significant carry-over effects, P = 0.3 and P = 0.1, or time effects, P = 0.7 and P = 0.7, for either creatinine clearance or p-potassium.Five patients had hypoalbuminemia, with a p-albumin <3.6 g/dL. There was a significantly higher concentration of albumin in plasma during treatment with eplerenone, P = 0.004. When the hypoalbuminemic patients were taken out of the statistical analysis, the P-value changed to 0.006. Twenty-nine patients had higher mean values of p-albumin during the treatment period, and 10 patients had lower values. Only one of the hypoalbuminemic patients had normalization of p- albumin in the treatment period and a 17% reduced urinary albumin excretion in the treatment period. Four patients were continuously hypoalbuminemic throughout the study. Two of these patients had a reduced excretion of albumin in urine and two had a higher excretion during treatment with eplerenone. Along with the analysis of changes in p-albumin it was also investigated whether there were any changes in p-cholesterol , p-LDL and p-HDL cholesterol and triglycerides. Neither of these changed significantly during the study. No patients were started or altered in lipid lowering treatment during the study period.There were also no significant differences between study periods in the following parameters: weight, BMI, p-ionized calcium, p-uric acid, p-C-reactive-protein, blood haemoglobin, nor in the urinary excretion of creatinine, potassium and sodium.The present study is the first to demonstrate an antiproteinuric effect of the selective aldosterone receptor antagonist, eplerenone, on overt albuminuria in non-diabetic CKD. Thus, in 40 CKD patients, add-on of eplerenone to stable antihypertensive treatment including RAS-blockade for eight weeks caused a 22% reduced excretion of urinary albumin compared to a control period. BP and creatinine clearance were slightly lower during the eplerenone period, but even after correction for that the urinary albumin excretion was significantly lower during treatment. The validity of the present observation rests on the randomized design of the study. The cross-over design was chosen in order to minimize the influence of heterogeneity between subjects and the lack of placebo control. The absence of a carry-over effect from eplerenone to control confirmed that no wash-out period after eplerenone was necessary.Reduction in albuminuria during aldosterone blockade in the present study was less than that observed in the earlier studies. Despite this, it is relevant to consider addition of eplerenone in CKD patients on RAS-blockade, due to the fact that residual proteinuria influences the prognosis for proteinuric CKD The antiproteinuric effect of eplerenone has previously only been reported in patients with type 2 diabetes with microalbuminuria and in patients with essential hypertension Studies with spironolactone have shown reduction in urinary protein excretion in the order of 30–58% in patients with diabetic and non-diabetic CKD stage 1–2 There may be two explanations for the antialbuminuric effect of aldosterone antagonism being smaller in our study than in the previous ones: First, in our study albuminuria was monitored in repetitive 24 hour urine samples collected for every visit and subsequently averaged, aiming at a high degree of accuracy of results. By contrast, some previous studies were based on a limited number of UACR measurements The present study was not powered to detect differences between groups with different intensity of baseline RAS-blockade. Patients were on stable RAS-blockade in a dose at the discretion of their treating physician. In a post-hoc analysis we found that the reduction in proteinuria during add-on eplerenone tended to be most pronounced in patients treated at baseline with a moderate level dose of RAS-blockade. In a recently published study of diabetic patients with a UACR≥300 mg/g creatinine treatment was initially a supramaximal dose of ACEi. Spironolactone or an ARB was added for 48 weeks. Reduction in UACR was seen in both groups, but largest in the spironolactone group Interestingly, an antiproteinuric effect of eplerenone was found in two out of the three patients with membranous nephropathy, a disease shown in a previous prospective study to be resistant to aldosterone inhibition As shown in A short-term study of aldosterone blockade with spironolactone in CKD has likewise shown an initial decrease in albuminuria after just two weeks of treatment P-potassium values rose during the treatment period. The mechanisms involved in the antialbuminuric effect of aldosterone receptor blockade have not yet been fully elucidated. At present it is not known whether it is the classical or the non-classical (genomic or non-genomic) effects of aldosterone that influence progression of renal damage. An in vitro study has shown that eplerenone blocks the non-genomic pathways of aldosterone The potential risks of blocking aldosterone receptors are mainly hypotension and hyperkalaemia. Previous studies initially reported no major clinical problems with hyperkalaemia Eplerenone in contrast to spironolactone does not cause gynecomastia or breast tenderness Hence, eplerenone may be a useful drug in proteinuric patients with CKD. The present study contributes to demonstrate eplerenone as an alternative to treatment with spironolactone and as a treatment alternative to patients who do not tolerate ACE-inhibition due to side effects.The group of patients studied were not homogeneous with respect to baseline albuminuria and renal diagnoses. There were no requirements to baseline therapy or run-in period. The study was not designed to detect a difference in response to eplerenone based on the baseline RAS-blocking treatment. It was an open label design and a relatively small sample size, which limits the extent to which results should be generalized. The statistical analyses were based on samples from all visits which were given equal weight in the analyses.Eplerenone in a dose of 25–50 mg daily is a safe and relevant add-on therapy in patients with non-diabetic CKD when aiming at a reduction in urinary albumin excretion. The potential risk of hyperkalaemia, especially in daily practice outside the setting of a controlled trial, should be kept in mind. Long-term studies need to be designed in order to assess the question of whether the effect of eplerenone on the reduction in urinary albumin excretion is persistent, and whether the progression rate of CKD is reduced.Checklist S1An index of where in the article specific parts of the required information may be found.(DOC)Click here for additional data file.Protocol S1The study was carried out according to the protocol, which can be accessed here.(DOC)Click here for additional data file.


The analyses showed that tissue is an important variability factor in miRNAs expression stability whereas breed is not a determinant factor. All ten miRNAs analysed had good stability values and, therefore, can be used as reference miRNAs. When all tissues were considered, miR-93 was the most stable miRNA. Dividing data set by tissues, let-7a was the most stable in skeletal muscle and ovary, miR-17-5p in kidney, miR-26a in liver and miR-103 in uterus. Moreover, the optimal number of reference miRNAs to be used for proper normalisation data was determined. It is suggested the use of five reference miRNAs  in multi-tissue experimental designs and the use of three reference miRNAs as the optimal number in single tissues studies . Overall, this study provides valuable information about the porcine reference miRNAs that can be used in order to perform a proper normalisation when relative quantification by RT-qPCR studies is undertaken.Relative quantification is the strategy of choice for processing RT-qPCR data in microRNAs (miRNAs) expression studies. Normalisation of relative quantification data is performed by using reference genes. In livestock species, such as pigs, the determination of reference miRNAs and the optimal number of them has not been widely studied. In this study, the stability of ten miRNAs  was investigated by RT-qPCR in different tissues  and in different pig breeds  as variation factors. Stability values were calculated with geNorm and NormFinder algorithms obtaining high correlation between them (r MicroRNAs (miRNAs) are small non-coding RNAs involved in gene expression regulation at the post-transcriptional level in animals, plants and viruses Northern blot has been widely used for determining and measuring miRNA expression One of the most extensive strategies used to evaluate and compare RT-qPCR data is relative quantification In miRNA expression studies, the most common reference genes used are ribosomal RNAs, such as 5S RNA The aim of this study was to analyse the miRNA expression stability in different porcine tissues and breeds. Selected tissues were skeletal muscle , uterus and ovary (reproductive tissues), liver (metabolic tissue) and kidney (excretory tissue). On the other hand, porcine breeds include Iberian (European breed), Meishan and Vietnamese (Asian breeds) and Landrace and Large White . The results from this work provide useful information concerning which miRNAs could be effectively used as reference genes in order to measure miRNA expression accurately through RT-qPCR studies.In accordance with the most stable miRNAs described in the literature 2 = 0.99, Firstly, the stability was evaluated taking into account the entire data  with geNorm Although all miRNAs showed good stability values, the most stable miRNA was miR-93, followed by miR-25, miR-106a, miR-17-5p and miR-26a . InteresIt is known that the stability of miRNA expression could change when factors like tissue or breed are considered. Thus, the entire data were divided by breeds. The results obtained did not differ greatly compared to when the entire data were analysed. M and SV values were calculated from 0.64 to 0.90 and from 0.22 to 0.53, respectively (data not shown). The most stable miRNAs were still ranging between those most stable in the general study. miR-93 remained the most stable miRNA in Iberian and Meishan breeds, miR-26a in Landrace and Vietnamese breeds and miR-25 in Large White breed. The least stable miRNAs were miR-191 in Iberian and Landrace breeds, miR-16 in Large White and Vietnamese breeds and miR-26a in Meishan breed (data not shown). Overall, our results showed that breed only slightly influences the stability of miRNAs.The stability of the miRNAs was also evaluated for each of the five tissues analysed. As expected, the stability of the miRNAs varies among tissues . M and SComparing the stability values obtained in the entire data with those obtained in each tissue group, the M and SV values generally improved when tissues were treated as separated . This isIt is important to take into consideration the physiological status  of the sows because it is known that hormones could affect the gene expression. However, the ten reference miRNAs tested in this study showed no significant differences in miRNA expression between pre and post-pubertal sows. Then, it seems that the expression of these 10 miRNAs in the studied samples is stable under different hormonal environments.The optimal number and choice of reference miRNAs for qPCR data normalisation must be experimentally determined. Moreover, more than one reference miRNA should be used In studies considering only one tissue, the use of three reference miRNAs would be optimal, taking into account that the V-values using three reference miRNAs were below 0.10 in all tissues . To contIn conclusion, this work has evaluated the stability of ten miRNAs in different porcine tissues and breeds showing that they could be used as reference miRNAs. Stability values reflect that tissue is an important variability factor and it must be taken into consideration in the experimental design. It is recommended the use of five reference miRNAs: miR-93, miR-25, miR-106a, miR-17-5p and miR-26a in studies which include multiple tissues. For studies in a specific tissue, the optimal would be the use of three reference miRNAs which is sufficient to obtain a reliable normalisation of data. The most stable reference miRNAs vary between the tissues studied. In kidney it is recommended miR-17-5p, miR-93 and miR-26a. In ovary the best options are Let-7a, miR-25 and miR-106a, while in uterus we recommend to use miR-103, Let-7a and miR-93. If the study is focused in skeletal muscle, we encourage using Let-7a, miR-17-5p and miR-25, but if we are working with liver, the most stable miRNAs to be used as reference miRNAs are miR-26a, miR-103 and Let-7a.Overall, this study provides valuable information about the porcine reference miRNAs that can be used in order to perform a proper normalisation when relative quantification studies are undertaken. Further experiments should be made to construct a database with recommended reference miRNAs for each tissue in porcine and also for multiple tissue studies.Samples were collected from five different pig tissues . Pigs included in the study came from different breeds: Iberian (IB), Meishan (ME), Vietnamese (VT), Landrace (LD) and Large White (LW). Two samples per breed and per tissue were analysed (n = 50). All animals were females in different physiological state  and age . All samples were taken from slaughterhouse  under veterinary supervision. Samples were immediately snap-frozen in liquid nitrogen and stored at -80°C until use.Ten miRNAs were selected to be evaluated as reference genes according to the literature: Ssc-let-7a, Ssc-miR-103, Ssc-miR-17-3p, Hsa-miR-25, Hsa-miR-93, Ssc-miR-106a, Ssc-miR-191, Ssc-miR-16, Ssc-miR-26a and Ssc-miR-17-5p Total RNA was isolated using TRIzol® reagent following the manufacturer’s recommendations . RNA was quantified by absorbance using ND 1000 Nanodrop® Spectrophotometer  and checked for integrity by using RNA 600 Nano kits  on an Agilent 2100 Bioanalyzer.Reverse transcription (RT) reactions were performed in duplicate using total RNA as previously described Quantitative PCR reactions were performed in a final volume of 20 µL including 10 µL of FastStart Universal SYBR Green Master , 250 nM of each primer  and 5 µL of a 1∶2000 dilution of the cDNA (except for Ssc-miR-17-3p where a dilution of 1∶200 was used). See Standard curves were generated in order to calculate the RT-qPCR efficiency. All standard curves were done by using 10 fold serial dilutions from a pool of cDNA of all the samples (n = 50) and were included per duplicate in all qPCR assays. For Ssc-miR-17-3p, a 2-fold dilution standard curve was used. Reactions were incubated in a 96-well plate at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min on a 7900HT Real-Time PCR System with 7900HT SDS v2.4 (Applied Biosystems). DNA primers for each miRNA were designed following the methodology described by Measurements were performed in duplicate. Minus RT controls, minus poly(A) polymerase controls and no template controls were included. Moreover, melting curve analysis was performed in each assay in order to detect unspecific amplifications.Quantities from each sample were obtained from the calibration (standard) curve added in each RT-qPCR reaction. Stability of each candidate miRNA was tested using geNorm v.3.5 algorithm 2 transformed and significance threshold was set at α<0.05. Student-Newman-Keuls (SNK) and Scheffe tests were used to determine significant differential expression between breed or tissue groups.RT-qPCR expression data was also analysed by a two-way analysis of variance (ANOVA) with the General Linear Models procedure of the Statistical Package for the Social Scientists . The model used included breed and tissue as fixed factors and breed by tissue interaction. GeNorm v.3.5 software


A community-based life style is the normal mode of growth and survival for many bacterial species. These cellular accretions or biofilms are initiated upon recognition of solid phases by cell surface exposed adhesive moieties. Further cell-cell interactions, cell signalling and bacterial replication leads to the establishment of dense populations encapsulated in a mainly self-produced extracellular matrix; this comprises a complex mixture of macromolecules. These fascinating architectures protect the inhabitants from radiation damage, dehydration, pH fluctuations and antimicrobial compounds. As such they can cause bacterial persistence in disease and problems in industrial applications. In this review we discuss the current understandings of these initial biofilm-forming processes based on structural data. We also briefly describe latter biofilm maturation and dispersal events, which although lack high-resolution insights, are the present focus for many structural biologists working in this field. Finally we give an overview of modern techniques aimed at preventing and disrupting problem biofilms. Furthermore, communication between sibling and rival cells has led to the evolution of multicellular, higher ordered life. For many years it was believed that bacteria inhabit the planet exclusively in a planktonic form, as free-living cells, but it is now widely accepted that most microorganisms reside primarily in biofilms -4. A bioBacterial biofilms are ubiquitous in the environment and can be found on almost any hydrated non-shedding surface; including rivers, stagnant pools, man-made materials and biological matter. Whilst many of these communities can be used for the synthesis of valuable products and the treatment of waste, others can cause problems in industrial applications and/or persistent virulence in humans and other animals. These bacterial aggregates provide protection from a wide range of environmental factors such as fluctuating pH , exposur1) , reesponses ,36 and cesponses . Bap is esponses  and also pathway .S. aureus biofilm formation via attachment to the ECM. These include the fibrinogen binding proteins: clumping factor A (ClfA) and ClfB (~95 kDa) [Spa and FnBPs belong to a large family of proteins called MSCRAMMs   that ena~95 kDa) -43; and ~95 kDa) ,36,44. M~95 kDa) ,42,45. 2A). The repeat/non-repeat region can be further divided into an A-region and B-region. The A-region contains ligand binding domains (N1-N3) whilst the role of the B-region is to project the adhesive domains away from the bacterial surface, although they also contain unknown functions. In FnBPs, the B-region in addition harbours the fibronectin binding activity. MSCRAMMs are modular in nature and contain an N-terminal signal sequence followed by an extensive repeat/non-repeat region and a C-terminal region for cell wall anchoring   [S. aureus metalloprotease autolysin [E. coli have unstructured N1 domains that are degraded by endogenous proteases prior to purification [2B). Each domain is dominated by β-sheet secondary structure that folds into a variation of the IgG fold. This DEv-IgG fold, named so because two additional strands (D’ and D’’) are inserted between the D and E strands of the standard IgG fold, was first identified in the collagen binding MSCRAMM, Can (pdb: 1AMX) [The B-region of ClfA/B is composed of mainly serine and aspartate residues (R-region) and it has been shown that residues 229-545 of ClfA  are sufficient to retain the binding activity for the C-terminal region of the fibrinogen γ-chain (Krimetry) . The N1 utolysin  and recofication . The cryfication  with theb: 1AMX) , but hasb: 1AMX) ,53. Cna b: 1AMX) . 2 of surface area. Ganesh et al. [In the crystal structure of ClfA in complex with a 13 residue fibrinogen-derived peptide (pdb: 2VR3), the ligand is recognized and binds along the interface between the N2 and N3 domains . Comparih et al.  have alsStreptococcus dysgalactiae FnBP in complex with the fibronectin module pair 1F12F1 (pdb: 1O9A) and showed how these class of proteins interact with fibronectin through a tandem β-zipper mechanism [2F13F14F15F1  [2C), again as a tandem β-zipper. Although these interactions are solely mediated by β-sheet interactions, there is ~4300 Å2 of surface area buried in each of the FnBR/2F13F14F15F1 complexes. It is likely that the interaction with fibronectin is multivalent, binding six to nine fibronectin copies per FnBPA/B molecule [2 [The A-region of FnBPA/B can also bind the C-terminal region of the fibrinogen γ-chain and likely recognizes this ligand in a similar fashion to ClfA/B ,56. The echanism . More reY, 2RKZ) . Each Fnmolecule ,61 and tecule [2 . 2.2Streptococcus parasanguis FW213 [Fimbriae-associated protein 1 (Fap1) was identified by Paula Fives-Taylor’s group whilst studying novel adhesive proteins from the Gram-positive is FW213 . Sanguisis FW213 . Furtheris FW213 . In addiis FW213 ,65-67.Staphylococcus aureus SraP [Staphylococcus agalactiae Srr-1 and Srr-2 [Streptococcus gordonii GspB [S. gordonii Hsa [Staphylococcus saprophyticus UafB [Streptococcus pneumoniae PsrP [ Streptococcus sanguinis SrpA [3A).Fap1 is a ~200 kDa surface fibre which is essential for fimbrial biogenesis, adhesion and biofilm formation ,67-70. Aeus SraP , Staphylnd Srr-2 -80, Strenii GspB ,81, S. gonii Hsa , Staphylcus UafB , Streptoiae PsrP -87, and nis SrpA . Primary3A) [SRRPs are glycosylated in the cytoplasm ,89-91 be3A) ,93,94. Iin vitro tooth model: saliva-coated hydroxylapatite (SHA) [α) followed by a β-sheet (Fap1-NRβ) region. NMR analysis and SHA assays show that whilst both are involved in binding to a yet unknown host ligand in the salivary pellicle, it is the helical subdomain that directs the pH-mediated effects [3B). Comparison of the crystal structure of Fap1-NRα at pH 5.0 [β into these low resolution envelopes identifies a mechanism of adhesion modulated by electrostatics  [The ‘non-repeat’ region of Fap1 (Fap1-NR) was characterized as an adhesive domain after it was shown to interact with an te (SHA) . Furtherte (SHA) . Fap1-NR effects . SAXS dat pH 5.0 ,94 and tt pH 5.0  shows noFig. 3B) ,93. 3B) [S. parasanguis does not have an acid tolerance response [S. parasanguis under metabolically active neutral/alkali conditions, but at low pH whilst metabolically dormant, Fap1 provides a much higher affinity for the salivary pellicle and in turn the mature biofilm which protects S. parasanguis [In Fap1-NR Asp152, Glu154, Asp430 and Asp435 pack against one another within the inter-subdomain face and this has the effect of raising the local pKa of these carboxylate groups so that they are protonated at a higher pH  . These s3B) . Thereforesponse ,97 and iresponse . So Fap1asanguis . Streptococcus pneumoniae, SraP from Staphylococcus aureus and GspB from Streptococcus gordonii strain M99 can all mediate inter-bacterial biofilm interactions in the lungs of infected animals via their unique non-repeat regions, in addition to the well documented host adhesive properties [BR) from S. gordonii was solved  in Tina Iverson’s group, and this has exemplified how the basic SRRP architecture can accommodate a plethora of different functions [S. gordonii M99 and loss of expression leads to a noticeable effect on the pathogenicity of this strain [Work in Carlos Orihuela’s group has shown that the SRRPs PsrP from operties . Recentlunctions . GspB iss strain  and specs strain . BR is composed of three adjacent domains and has been described as being like beads on a string [S. aureus Cna [S. parasanguinis Fap1-NRβ [BR in complex with an analogue of sialyl-T antigen confirmed this function . A deviation of the subdomain orientation within these two GspBBR structures suggests that these regions display independent motion. Furthermore, the modular nature of these motifs hints at multiple functions, which may be independent or cooperative as within Fap1-NR [BR cause a significant reduction in bacterial densities within kidneys and spleens and so it is likely that it is this region that facilitates the main S. gordonii M99 interactions in endocarditis [The crystal structure of apo-GspBa string . The N-treus Cna  and S. pFap1-NRβ . Whilst  Fap1-NR . Mutatiocarditis . However3S. aureus SasG  from Jennifer Potts group gives a tantalizing suggestion of how these classes of proteins may form protein-protein or protein-saccharide/ DNA interactions through the sequestration of Zn2+ [Once prokaryotes have formed a tight adherence to a surface they begin to divide and scan their local environment for interactions with siblings and genetically distinct partners. This marks the initiation of the three-dimensional architecture of a biofilm. At this stage cellular communications are mediated by surface exposed protein interactions and while our biochemical and genetic understanding of these processes are quite detailed, high resolution structural information is lacking. Again in this section, structures will only be described which allow an atomic understanding of early biofilm formation. A recent crystal structure of the B-region of  of Zn2+ , yet as 3.1Escherichia coli and recently Joseph St Geme’s group has published the structure of Hap from Haemophilus influenza (pdb: 3SYJ) and presented a general model for SAAT-mediated bacterial auto-aggregation [Autotransporters (ATs) belong to the type V secretion system family and account for the largest number of exported proteins from Gram-negative bacteria . ATs conregation ,109. H. influenza is an agent of bacteremia, pneumonia and acute bacterial meningitis. Hap is involved in the adhesion of H. influenza to epithelial cells and extracellular matrix proteins, invasion of epithelial cells and inter-bacterial aggregation during early biofilm development [s) consisting of a serine protease, an ECM-binding/SAAT domain and finally the C-terminus comprises of the outer membrane β-barrel motif . The ECM-binding domain is responsible for recognizing fibronectin, laminin and collagen IV [4A). The main spine of the structure is formed from a β-helix and has at the C-terminus the SAAT domain, overlapped with the ECM-binding domain. The N-terminal region of the β-helix acts as a scaffold for the positioning of the serine protease at the extreme N-terminus of the passenger domain.elopment . The N-tlagen IV  and the lagen IV  and alsolagen IV ,113. Thetrans configuration by a crystallographic 2-fold screw axis . Inter-dimer interactions are mediated in the main by a ladder of Asn/Asp hydrogen bonds from one monomer to the face of another, with also some burial of hydrophobicity. A secondary dimer interface is formed by a region flanking either end of the SAAT domain. As this dimer forms with a single monomer’s Asn/Asp ladder, the other remains exposed for higher ordered oligomerization. Further symmetry relations build an array of multimerized Hap molecules with the N-terminal β-helix facilitating significant inter-molecular interactions . These inter-Hap mediated interfaces have been rigorously tested using a number of mutants in a self-association assay and this presents a valid model for Hap-Hap biofilm activity. Once this initial dimer has formed in vivo it likely acts as a nucleant for the assembly of mega-Dalton complexes of immense stability that overcomes the repulsive force between bacteria [The SAAT domain of Hap is entirely β-helical and displays a hydrophilic edge of stacked Asn/Asp residues . Furtherbacteria . 4C) [H. influenza surface, whilst in its presence this activity is revoked resulting in oligomerization and inter-bacterial aggregation. In the work by Meng and colleagues , they we4C) . This su3.2 Escherichia coli associated with newborn meningitis and septicaemia (NMEC) [E. coli strains and it is now usually referred to as the E. coli common pilus (ECP) [E. coli are primarily commensal colonizers of the human and other animal bowels and they contribute to a healthy immune system of the host. There are also a number of virulent strains that can cause diarrheal diseases such as hemorrhagic colitis [In 2001 Timo Korhonen’s group published data on a novel fimbria isolated at low temperatures froma (NMEC) . This waus (ECP) -119. E.  colitis . Further colitis .5A) [ECP fibres are assembled via a variant of the CU pathway and as with all members of this superfamily, filaments are formed from polymerisation of several different/identical pilin domains  ,122. The5A) ,116, whi5A) .E. coli (UPEC) has been recently solved by our group [5B). EcpA is fashioned from approximately 50% hydrophobic residues and the surface is scattered with hydrophobic patches including a number of aromatic residues. This likely promotes a less-specific contact with a wide range of hydrophobic substrates and polymers. The crystal structure of EcpA  from uropathogenic ur group . Like ot5C) [Pili assembled via the CU pathway vary greatly in size and function. Afa-III fimbrils are very flexible and are composed of a head-to-tail polymerisation of subunits ~2 nm in diameter , whilst 5C) .E. coli producing ECP show these fibres form a mesh that encapsulates the whole microcolony. ECP interacts with itself through pili crossing over one another, parallel fibre entwining and antiparallel entwining [5D). Inter-ECP interactions are conducted in the main via burial of the loop residues Ala147, Val148 and Thr149 across the central axis and mutations of these residues to bulky amino acids results in dramatic reductions in cell mass in biofilm assays. EcpA is highly conserved amongst a range of other enteric bacterial species including Serratia proteamaculans, Serratia odorifera, Klebsiella sp., Klebsiella pneumoniae, and Enterobacter cancerogenus, which suggests a role for ECP in establishing contacts between multiple species [EM images of ntwining ,122. The3.3S. parasanguis when experiencing long periods of acidity. Moreover, there is much evidence that Fap1 also functions in mid and later stages of biofilm development. The heavy glycosylation has been implicated in some of these processes [S. parasanguis using EM also shows a pH-dependence to the auto-aggregation of these fibres, specifically at their N-terminal pole [S. parasanguis over a range of pH values show a clear increase in cellular mass correlated to acidity, mirroring the binding of Fap1-NR to SHA [Fap1 is a model system to study the biogenesis, export and general architecture of SRRPs; however, in its own right it displays some very unique properties. As has been detailed above, the binding of Fap1 to SHA is affected greatly by the pH of the environment and this can be attributed to a survival mechanism of rocesses  and the nal pole . In addiR to SHA ,93.α has been very insightful in terms of our understanding of how Fap1 may form inter-filamentous aggregation via the N-terminal tip  [α in the crystal lattice clearly demonstrates an order that can accommodate full Fap1-NR domains and allow the major SRR region to project back to a bacterial surface from multiple orientations [6B). This is consistent with the observations that Fap1 tip interactions can form at pH 8.0 but become much more prevalent at pH 5.0 [The crystal structure of Fap1-NRFig. 6A) . The arrntations , in a sintations . No intet pH 5.0 . Further4In the middle stages of biofilm formation, cells develop into a three-dimensional array and start producing the sticky conglomerate EPS, of which the composition can be very diverse between different species. On average 90% of a biofilms dry mass will be accounted for by the EPS, and this is the immediate environment which these prokaryotes sample . BiofilmPseudomonas aeruginosa [Myxococcus xanthus were observed under EM [Thiobacillus ferrooxidans to pyrite surfaces [The major contributors of the bacterial EPS matrix are polysaccharides  and manyruginosa . Dependiunder EM . Some bisurfaces S. aureus it is a major component of the matrix structure, it is less so in S. epidermis [Enterococcus faecalis are able to specifically lyse a fraction of the bacterial population using a protein GelE [P. aeruginosa eDNA is used to connect cells and treatment with DNase inhibits biofilm formation [Bacillus cereus eDNA has also been shown to function as an adhesin [P. aeruginosa [Nucleic acids in the form of DNA can be in abundance in biofilms, although whilst in pidermis . The souein GelE . In P. aormation ,138, whi adhesin . Furtherruginosa , resultiE. coli and Salmonella enterica serovars Typhimurium biofilm matrix. Curli are crucial in these biofilms and mediate initial surface attachment and provide a scaffold for the community [Another important type of macromolecule involved in the maturation and shaping of biofilms are microbial amyloid fibres . Amyloidommunity ,146,147.ommunity , the mol5The controlled restructuring of biofilm architecture results in a heterogeneous arrangement of matter and the production of cavities and channels. This process can be undertaken by both targeted cell lysis and cell dispersion. Another process that is closely linked to these restructuring events is the partial breakdown of the matrix to allow the release of cells, which are free to migrate and inhabit new environments, i.e. when nutrients become limiting and when waste products accumulate . These eAggregatibacter actinomycetemcomitans is a Gram-negative non-motile commensal of the oral cavity, associated with gum disease. Dispersin B is an extracellular enzyme (PDB: 1YHT) [A. actinomycetemcomitans and can degrade matrix polysaccharides [A. actinomycetemcomitans biofilms [N-acetylglucosamine substrate [B: 1YHT)  secretedcharides . This isbiofilms , it has ubstrate ,159. P. aeruginosa rhamnolipid [S. aureus biofilm maturation process [An alternative strategy of restructuring/dispersal is exemplified by the mnolipid ,160 and mnolipid ,161. Themnolipid -164 and mnolipid . These a process  includin6Biofilms represent the dominant microbial life style in aquatic environments, providing nutrients, purification of water, sequestration of carbon and biogeochemical fluxes; i.e. roles that are essential for these and other habitats to exist . CompounS. aureus and P. aeruginosa [One point of entry to combat biofilms is to stop them before they can take hold through inhibition of the initial attachment. A number of strategies have been investigated that target both the adhesive mechanisms and also the biogenesis pathways from which these adhesins are produced. An example of the latter are Gram-negative bacteria that use CU assembled pili as adhesive filaments: compounds termed ‘pilicides’ have been shown to disrupt the biogenesis machinery of the type I pilus and inhibit biofilm formation in UPEC ,168. Antruginosa . It is aruginosa ,171 and ruginosa  both dicOnce a biofilm has become established, modern antibiotics have little effect on their displacement  and can Bacillus licheniformis lipopeptide biosurfactant has been shown to have a marked effect on increasing antibiotic activity against E. coli biofilms [Bacillus subtillis release a factor that inhibits the formation of, and initiates the breakdown of existing biofilms [S. aureus and P. aeruginosa biofilms. These amino acids function through incorporation into the cell wall peptidoglycan where they stimulate release of the amyloid TasA from the matrix [Bacillus subtillis compound, norspermidine, seems to act in a complimentary manner by targeting exopolysaccharides [Enzymes perform functions in biofilm restructuring and whilst they are highly specific, they are expensive to produce and can be unstable. Furthermore, in a medical setting they may also cause an unwanted immune response, therefore the direct use of enzymes in therapeutics does not seem viable. Phages, however, have been reported to improve the normal antimicrobial activity in biofilm related infections  and morebiofilms . Althougbiofilms . This wae matrix ,181. In charides . S. aureus quorum-sensing inhibits biofilm development [agr (accessory gene regulator) locus, which produces and senses AIP (autoinducing peptide). AIP is an eight residue peptide where the C-terminal five residues form a cyclic thiolactone ring [P. aeruginosa produce an organic compound, cis-2-decenoic acid, which can disseminate established biofilms and also inhibit biofilm development [P. aeruginosa this highly potent fatty acid can also disrupt many other Gram-negative biofilms. A number of these natural dispersal mechanisms have been coordinated with current antibiotic therapies and have shown great promise in dissemination of medically relevant biofilms [Bacteria have developed highly robust cell-cell signalling or quorum-sensing mechanisms. In elopment ,184 via one ring . AIP hasone ring . Furtherelopment . This mabiofilms . Biofilms represent the most frequent mode of growth for many microbes. While headway is being made in understanding their formation and development, we are still far from being able to describe all of these processes from a molecular perspective. As further insights into this complicated life style are made available, both at the atomic and cellular level, new targets to be exploited will arise, giving us a much wider scope to address problematic biofilms.


The purpose of this study was to clarify the influence of the thickness of the implanted DN gel on the induction ability of hyaline cartilage regeneration.A double-network (DN) gel, which is composed of poly(2-acrylamido-2-methylpropanesulfonic acid) and poly, can induce hyaline cartilage regeneration Thirty-eight mature rabbits were used in this study. We created an osteochondral defect having a diameter of 4.3-mm in the patellofemoral joint. The knees were randomly divided into 4 groups . Animals in each group were further divided into 3 sub-groups depending on the gel implant position  in the defect. The regenerated tissues were evaluated with the Wayne’s gross and histological grading scales and real time PCR analysis of the cartilage marker genes at 4 weeks.According to the total Wayne’s score, when the depth of the final vacant space was set at 2.0 mm, the scores in Groups I, II, and III were significantly greater than that Group IV (p < 0.05), although there were no significant differences between Groups I and IV at a 3.0-mm deep vacant space. The expression levels of type-2 collagen in Groups II and III were significantly higher (p < 0.05) than that in Group IV.The 1.0-mm thick DN gel sheet had the same ability to induce hyaline cartilage regeneration as the 5.0-mm thick DN gel plug. However, the induction ability of the 0.5-mm thick sheet was significantly lower when compared with the 1.0-mm thick gel sheet. The 1.0-mm DN gel sheet is a promising device to establish a cell-free cartilage regeneration strategy that minimizes bone loss from the gel implantation. It has been a common belief that the articular hyaline cartilage tissue cannot spontaneously regenerate in vivo,2. Therein vivo-10. Recein vivo-14. Howein vivo within only 4 weeks in the vacant space [in vivo cartilage regeneration, because it may affect biomechanical environment in the defect. Thus, we have conducted this in vivo study in order to clarify if the gel thickness influences on the in vivo hyaline cartilage regeneration induced by the DN gel implanted at the bottom of a large osteochondral defect.Recently, we have found that, when we implant a 5 to 8-mm thick PAMPS/PDMAAm double-network (DN) gel plug at the bottom of a large osteochondral defect created in the rabbit knee joint so that a 2- to 3-mm deep vacant space is intentionally left in the defect, cartilage regeneration can be induced nt space . This DNnt space ,17. HoweRecently, we have developed a DN gel sheet thinner than 1.0 mm with our advanced gel technology. In this study, we have hypothesized that a 1-mm thin PAMPS/PDMAAm DN gel sheet may be able to induce cartilage regeneration in an osteochondral defect, when it was implanted at the bottom of the defect so that a 2- to 3-mm deep vacant space was left in the defect, but that a 0.5-mm thin DN gel sheet may not be able to induce cartilage regeneration. The specific aim of this study was to test this hypothesis.The PAMPS/PDMAAm DN gel is a kind of interpenetrating network hydrogel, but with an asymmetric structure: In the DN gel, the two independently cross-linked polymer networks are physically entangled with each other. This DN gel is strong enough (the compressive fracture strength of 3.1 MPa ) to crea’-methylenebisacrylamide (MBAA) as a cross-linker and 2-oxoglutaric acid as an initiator. The monomer concentration was 1 mol/l for PAMPS, 4 mol% for the cross-linker, and 0.1 mol% for the initiator. Aqueous solution containing a monomer, cross-linker, and the initiator was bubbled with nitrogen for 30 minutes, and then injected into a cell consisting of a pair of glass plates separated by a silicone rubber. The cell was irradiated with a UV lamp (wave length 365 nm) for about 6 hours. The DN gel was synthesized by the sequential network formation technique (two-step method). The PAMPS hydrogel (1st network) was immersed in an aqueous solution of 2 mol/L DMAAm, containing 0.1 mol% MBAA, and 0.1 mol% 2-oxoglutaric acid for one day until reaching the equilibrium. The 2nd network (PDMAAm) was subsequently polymerized in the presence of the PAMPS hydrogel by irradiating UV for 6 hours between two plates of glasses. After polymerization, the PAMPS/PDMAAm DN gel was immersed in pure water for 1 week and the water was changed twice daily to remove any un-reacted materials. Using this principle method, we created 5.0-mm thick DN gel blocks and 1.0- and 0.5-mm thick DN gel sheets, and then, 4.5-mm diameter plugs or sheets were punched out from each block or sheet. Subsequently, we prepared 5.0-mm thick DN gel plugs as well as 1.0- and 0.5-mm thick DN gel sheets, all of which had a 4.5-mm diameter.The PAMPS/PDMAAm DN gel was synthesized using the previously reported two-step sequential polymerization method . BrieflyA total of 38 mature female Japanese white rabbits, 6 months old and weighing 3.6 ± 0.7 kg, were used in this study. Animal experiments were carried out in the Institute of Animal Experimentation, Hokkaido University School of Medicine under the Rules and Regulation of the Animal Care and Use Committee, Hokkaido University School of Medicine (Reference number: 08-0068).In the first experiment, we used 30 rabbits. The bilateral 60 knees of the 30 rabbits were randomly divided into 4 groups of 15 knees each Figure . In eachThe second experiment was conducted using the remaining 8 rabbits, based on the first study results. The 16 knees in 8 rabbits were randomly divided into 4 groups same manner as made in the first experiment and the final vacant space was set at 2.0-mm depth. All rabbits were sacrificed at 4 weeks, and real time polymerase chain reaction (PCR) analysis was performed , purified IgG, Fuji Chemical Industries Ltd, Toyama, Japan) was used as primary antibodies. Immunostaining was carried out according to the manufacturer’s instructions using the Envision immunostaining system . Finally, the sections were counterstained with hematoxylin. Histology was evaluated with the scoring system reported by Wayne et al. , which wTotal RNA was extracted from the tissues regenerated in the defect, using the RNeasy mini kit . RNA quality from each sample was assured by the A260/280 absorbance ratio. The RNA (100 ng) was reverse-transcribed into single strand cDNA using PrimeScript® RT reagent Kit . The RT reaction was carried out for 15 minutes at 37 degrees Celsius and then for 5 seconds at 85 degrees Celsius. All oligonucleotide primer sets were designed based upon the published mRNA sequence. The expected amplicon lengths ranged from 93 to 189 bp. The sequences of primers used in real time PCR analyses for rabbit regenerative tissues were as follows: type-2 collagen forward GACCATCAATGGCGGCTTC; reverse CACGCTGTTCTTGCAGTGGTAG. Aggrecan forward GCTACGACGCCATCTGCTAC; reverse GTCTGGACCGTGATGTCCTC. Sox9 forward AACGCCGAGCTCAGCAAGA; reverse TGGTACTTGTAGTCCGGGTGGTC. GAPDH forward CCCTCAATGACCACTTTGTGAA; reverse AGGCCATGTGGACCATGAG. The real time PCR was performed in Thermal Cycler Dice® TP800  by using SYBR® Premix Ex TaqTM . cDNA template (5 ng) was used for real time PCR in a final volume of 25 microliter. cDNA was amplified according to the following condition: 95 degrees Celsius for 5 sec and 60 degrees Celsius for 30 sec at 40 amplification cycles. Fluorescence changes were monitored with SYBR Green after every cycle. A dissociation curve analysis was performed (0.5 degrees Celsius /sec increase from 60 to 95 degrees Celsius with continuous fluorescence readings) at the end of cycles to ensure that single PCR products were obtained. The amplicon size and reaction specificity were confirmed by 2.5% agarose gel electrophoresis. The results were evaluated using the Thermal Cycler Dice® Real Time System software program . Glyceroaldehyde-3-phosphate dehydrogenase (GAPDH) primers were used to normalize samples.All data were described as the mean and standard deviation values. A commercially available software program  was used for statistical calculation. The mean value of the Wayne’s scores was statistically compared among groups using two-way analysis of variance (ANOVA). Regarding the gene expression of cartilage markers, one-way ANOVA was performed for each gene. The Fisher’s protected least significance difference test was used for post hoc multiple comparisons. The significance level was set at p = 0.05.In gross observation of the joint surface repair, the defect having 2.0-mm depth with the 0.5-mm (Group-I), 1.0-mm (Group-II), or 5.0-mm (Group-III) thick DN gel implantation was completely filled with a white opaque tissue, while the untreated defect (Group-IV: control) surface showed reddish opaque, patchy tissues Figure . The defIn histological observations Figure , the vacConcerning the gross appearance score, the ANOVA demonstrated that there was a significant difference not only on the effect of the group (p < 0.0001) but also on the effect of the depth of the final vacant space (p = 0.0018). The post-hoc test showed that Groups I, II, and III were significantly greater than that Group IV , and Group II was significantly greater than Group I p = 0.0354), when the depth of the final vacant space was set at 2.0 mm. Group III was significantly greater than Groups II and IV when the depth of the final vacant space was set at 4.0 mm . In Group II, the gross appearance score was significantly greater when the depth of the final vacant space was set at 2.0 mm and 3.0 mm than that of 4.0 mm   but also on the effect of the depth of the final vacant space (p = 0.0009). The post-hoc test showed that Groups I, II, and III were significantly greater than that Group IV , when the depth of the final vacant space was set at 2.0 mm. Groups II, and III were significantly greater than that Group IV , when the depth of the final vacant space was set at 3.0 mm, while there was no significant difference between Groups I and IV. There were no significant differences among the groups at 4.0 mm. In Group II, the histology score was significantly greater when the depth of the final vacant space was set at 2.0 mm and 3.0 mm than that at 4.0 mm . In Group III, the histology score was significantly greater when the depth of the final vacant space was set at 2.0 mm and 3.0 mm than that at 4.0 mm   but also on the effect of the depth of the final vacant space (p = 0.0004). The post-hoc test showed that Groups I, II, and III were significantly greater than that Group IV , when the depth of the final vacant space was set at 2.0 mm. Groups II and III were significantly greater than that Group IV , when the depth of the final vacant space was set at 3.0 mm, while there was no significant difference between Groups I and IV. At 4.0 mm, only Group III was significantly greater than Group IV p = 0.0059). In Group I, the total score was significantly greater when the depth of the final vacant space was set at 2.0 mm than that at 4.0 mm (p = 0.0206). In Group II, the total score was significantly greater when the depth of the final vacant space was set at 2.0 mm and 3.0 mm than that at 4.0 mm . In Group III, the total score was significantly greater when the depth of the final vacant space was set at 2.0 mm and 3.0 mm than that at 4.0 mm   and that in Group II was also significantly higher than that in Group I (p = 0.0473) Figure .in vivo hyaline cartilage regeneration phenomenon induced by DN gel is not influenced by the thickness of the gel implanted at the bottom of a large osteochondral defect, at least, in a range between 1.0 mm and 5.0 mm. Concerning the 0.5-mm thick sheet implantation, however, the gross observation score and expression level of type-2 collagen gene were slightly but significantly lower as compared with the 1.0-mm thick gel sheet implantation, while there were no significant differences in comparison with the 5.0-mm thick gel implantation. These results suggested that, when the thickness of the implanted gel is too thin, below 1.0 mm, the thickness of the implanted DN gel slightly but significantly reduces induction ability of the in vivo hyaline cartilage regeneration phenomenon.The present study demonstrated that, when each DN gel sheet or plug was implanted at the bottom of the defect so that a 2- to 3-mm deep vacant space was left in the defect, the evaluation scores were found to be the greatest. In this implantation condition, all the evaluation scores and the expression level of the type-2 collagen gene in the two groups with implantation of a 1.0-mm thick gel sheet or a 5.0-mm thick gel plug were significantly greater than those of the untreated control group, and there were no significant differences in those parameters between the two groups. These results suggested that the in vivo cartilage regeneration induced by the PAMPS/PDMAAm DN gel. Our previous in vitro study [in vivo cartilage regeneration induced by the PAMPS/PDMAAm DN gel has not been sufficiently clarified.We have studied the mechanism of the ro study  has showro study ,22. Recero study  reportedro study ,19 may ero study . Howeverin vivo hyaline cartilage regeneration, while the ability of the 0.5-mm thick DN gel implant was slightly but significantly inferior to that of the 1.0-mm thick DN gel implant. This fact implied that DN gel sheets thicker than 1.0 mm may provide the same physical environment under knee motion at the bottom of the defect with the 2- to 3-mm deep vacant space. Theoretically, however, when a very thin gel sheet is compressed by large forces, the apparent elastic modulus of the gel-bony floor (bone beneath the gel sheet) complex is increased by the effect of the extremely high modulus of the bony floor. Therefore, when the thickness of a DN gel sheet was 0.5 mm in this study, the apparent elastic modulus of the gel-bone complex might be too high under repetitive compression loading due to joint motion. Therefore, we speculate that the increased apparent modulus of the gel-bone complex under the repetitive compression loading may reduce the cartilage regeneration.The present study has added new information in order to increase the database on the hyaline cartilage regeneration induced by the PAMPS/PDMAAm DN gel. In the present study, first, the 1.0-mm and 5.0-mm thick DN gel implants had the same ability in the There are some limitations in this study. First, we used a rabbit patellofemoral joint model in this study, although this model has been commonly used to evaluate new procedures in cartilage regeneration experiments -27. The Concerning the safety of the PAMPS/PDMAAm DN gel as a biomaterial, we previously performed a pellet implantation test into the para-vertebral muscle , accordiAs to clinical relevance, the innovative strategy with the DN gel implantation for cartilage repair without cell culture has been proposed ,16. We sThe 1.0-mm thick DN gel sheet had the same ability to induce hyaline cartilage regeneration as the 5.0-mm thick DN gel plug. However, the induction ability of the 0.5-mm thick sheet was significantly lower when compared with the 1.0-mm thick gel sheet. The 1.0-mm DN gel sheet composed of PAMPS and PDMAAm is a promising device to establish a cell-free cartilage regeneration strategy that minimizes bone loss from the gel implantation.We have no financial or non- financial competing interests. We do not hold or are not currently applying for any patents relating to the content of the manuscript.HM performed animal experiments and PCR analysis. NK and KY designed the study, participated in the study, and drafted the manuscript. NK, KA, and FK participated in designing the study and instructed animal experiments. TK and JPG created the DN-gel material. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/14/50/prepub


Population data on mortality and life expectancy are generally available for most countries. However, no longitudinal data based on the health-related quality of life outcome from the EQ-5D instrument have been reported for orthopedic patients. We assessed the effect of orthopedic surgery as measured by EQ-5D. We analyzed EQ-5D data from 2,444 patients who were operated at the Department of Orthopedic Surgery at Karolinska University Hospital, 2001–2005. We also made a comparison between results from this cohort and those from a Swedish EQ-5D population survey.index score improved from 0.54 to 0.72. Hip and knee arthroplasty, operations related to previous surgery, trauma-related procedures, and rheumatoid arthritis surgeries had preoperative EQ-5D index scores of 0.48 to 0.52. All of these groups showed substantial improvement in scores (0.63 to 0.80). Patients with tumors or diseases of the elbow/hand showed higher preoperative scores (0.66 to 0.77), which were similar postoperatively. In most patients, the EQ-5D index score improved but did not reach the level reported for an age- and sex-matched population sample (mean difference = 0.11). The mean EQ-5D  Our results can be used as part of the preoperative patient information to increase the level of patient awareness and cooperation, and to facilitate rehabilitation. In future it will be possible—but not easy—to use the EQ-5D instrument as a complementary consideration in clinical priority assessment. Musculoskeletal conditions are the leading cause of severe long-term pain and disability in the world, affecting hundreds of millions of people . They arImprovement in health-related quality of life (HRQOL) is one of the most important goals of orthopedic surgery , a measure frequently used in cost-effectiveness analyses .index tariff). As there is no Swedish TTO tariff for EQ-5D health states, and since the only Swedish population survey to assess the EQ-5D used the UK tariff, we used the preference scores generated from the UK population when calculating the EQ-5D index scores for our study population. The patients completed the Swedish-translated questionnaire (EQ-5D 2009). By design, this descriptive system is able to identify 243 unique health states. An index score can be assigned to each of these health states to indicate its value or desirability from the point of view of the general public. Scores in the UK EQ-5D value set range from –0.594 for the worst possible health state to 1.0 for a perfect state of health, with 0 on the scale representing the state of being dead. Negative scores suggest that the corresponding health states are considered worse than being dead. Normally, the EQ-5D questionnaire needs 1 to 3 min for self-completion.Health-related quality of life data were obtained from the EQ-5D, a self-administered patient questionnaire . The EQ-index scores are reported as mean (SD). Age and sex standardized EQ-5D index scores at baseline (preoperatively and at 12 month follow-up (postoperatively) was calculated as the difference between observed scores and the age- (10 year age-groups) and sex-specific mean scores in the population survey. These preoperative and (12-month) postoperative EQ-5D index standardized scores are reported as mean (SD).The EQ-5D index score from baseline (preoperatively) and 12 months (postoperatively) were calculated and a paired t-test was used to test whether the change from baseline was equal to 0. We also analyzed the fraction of patients (by number and percentage) whose EQ-5D index score changed from baseline to 12 months (improved or deterioriated by > 0.1). Responders and non-responders at the 12-month follow-up were compared regarding age, sex, type of surgery, and preoperative EQ-5D by chi-square tests for qualitative variables and t-tests for quantitative variables. Even though the distribution of the change from baseline was not normal, the central limit theorem implies valid inference using the t-test when the sample size is more than about 30, and all but 1 subgroup had larger sample sizes. Since the fraction of responders was different for the different types of surgery, the comparisons with respect to age, sex, and preoperative EQ-5D score were also adjusted for this difference by analysis of variance and logistic regression.The changes in EQ-5D The final analysis included 2,444 patients, 57% of whom were women, and the mean age at surgery was 56 (SD 18) years . One thiindex score at baseline was 0.54, which is 0.29 units lower than would be expected in a population-based sample of the same age and sex distribution. On average, women had lower scores (0.50) than men (0.59) before surgery (p < 0.001), which remained unchanged after adjustment for age and type of surgery. Age did not affect the preoperative score substantially, with the exception of patients younger than 30 years, who had a higher mean score (by 0.12 units) than the average patients. This age effect could to some extent be explained by type of surgery. The different surgical procedures showed a wide spectrum of average EQ-5D index scores at baseline (0.30–0.77).The mean preoperative EQ-5D index score at baseline, procedures related to benign or malignant tumors and elbow/hand diseases scored statistically significantly higher than average, which is important clinically, while patients with hip and spine procedures scored significantly lower than average.When comparing the different surgical procedures for the overall mean EQ-5D index score (mean difference between the age- and sex-matched population) was –0.29. All 15 groups of patients had a lower EQ-5D index score than in the matched population (–0.09 to –0.53).Preoperatively, at baseline, the standardized EQ-5D index score had increased statistically significantly by 0.18 units from baseline to 0.72 . Patients with benign or malignant tumors or elbow/hand diseases showed no statistically significant changes in EQ-5D index score.Patients who underwent hip or knee arthroplasty, had complications after surgery, underwent other knee surgery, had trauma-related procedures, had rheumatoid arthritis or who underwent spine, hip, or infection-related surgery showed statistically significant improvements in mean EQ-5D index score (mean difference between the age- and gender-matched population) at 12 months of follow-up was –0.11. Hip arthroplasty patients had a mean standardized EQ-5D index score preoperatively of –0.31 but their EQ-5D score improved and reached the level of that of the age- and sex-matched population (standardized EQ-5D index score of 0.00 at 12-month follow-up). Knee arthroplasty, trauma-related operations, other hip and knee surgery, rheumatoid arthritis surgery, surgery after complications, and spine surgery showed major improvements in EQ-5D index score 12 months after operation. However, they did not reach that of the matched population. The mean difference in score from that of the matched population postoperatively varied from –0.07 to –0.21.The standardized EQ-5D index score and a small group (14 %) reported deterioration in their scores of > –0.1. 69% of the hip arthroplasty patients improved by at least 0.1 and only 6% deteriorated in their EQ-5D index score, in contrast to malignant tumor surgery where only 24% improved more than 0.1 and 30% deterioriated by > –0.1.One year after surgery, half of the patients experienced an improvement of > 0.1 in their EQ-5D index score was bimodal, and very few individuals scored around the average . However, after adjustment for type of surgery this difference was reduced to 3 years, but it was still highly significant (p < 0.001). A comparison between gender and response rate showed that women had a higher response rate . Adjustment of the association between gender and response rate for type of surgery reduced the association between gender and response rate . Similarly, after adjusting the difference in mean EQ-5D index score at baseline between responders and non-responders for type of surgery, the difference became less pronounced. On average, the responders had a lower score than non-responders by 0.05 units (p < 0.001). However, after adjustment for type of surgery, this difference was reduced to 0.03 units (p = 0.02).In the dropout analyses (Appendix) we found that the responders were more likely to be women, to be older, or to have a low preoperative EQ-5D index score increased from 0.54 to 0.72 one year after surgery.We found that most patients who were operated on for orthopedic conditions experienced an improved health-related quality of life and that their mean EQ-5D As expected, we noted large differences between surgical groups. In contrast to patients with tumor diseases, who scored high with a mean EQ-5D of 0.71, patients scheduled for hip or knee arthroplasty scored considerably lower . The indication for surgery is however, totally different in these cases, which must be kept in mind when interpreting these data. Notably, the group of patients treated with hip arthroplasty improved considerably and reached the scores of the age- and sex-matched reference population. Interestingly, patients with tumors improved in HRQOL to some extent in spite of their malignant conditions.In a review evaluating changes after hip replacement, the results from all studies were consistent in showing beneficial and often dramatic improvements in HRQOL after elective procedures . AnotherIn a study evaluating patients with inflammatory arthritis using both EQ-5D and SF-6D health assessment questionnaires, the authors recommended the inclusion of at least one preference-based measure in future clinical studies . We notiindex score has been reported by index score of 0.07 was found. In our orthopedic cohort, half of the patients had elevated EQ-5D index scores (by more than 0.1) after the operation. 14% had reduced EQ-5D index scores—by more than 0.1—one year after the operation, and one third had less changes (less than 0.1) in their EQ-5D index scores.The minimal important difference (MID) is important for interpreting the impact of score changes, and is also an important measure for power calculations in studies . MID forThis first attempt to collect a whole sample of orthopedic conditions makes it possible to perform cost-utility analysis. A QALY is defined as 1 year in full health. Estimation of QALYs requires data on survival and the corresponding health state score, the health status reflecting the HRQOL of the individual, on a scale from 0 (dead) to 1  . If utilIf baseline characteristics are controlled for the EQ-5D data, our findings could be used for comparison between hospitals. Comparison between provider units in different hospitals or between consultant specialities within a single institution can provide important information that might be applied for benchmarking or performance management.index score and two-thirds of them improved considerably. In future, “soft” HRQOL data (e.g. EQ-5D) might be included in the preoperative evaluation as well as more old-fashioned “hard” data such as radiology. However, to use the instrument in order to make priorities between groups of orthopedic surgical procedures seems to be more controversial, as the patients' individual EQ-5D index scores differed substantially.In addition to clinical priority assessment, criteria in elective orthopedic surgery EQ-5D could be used . PatientThe present study has several limitations. It is a prospective follow-up study of patients who underwent surgery, not a prospective randomized controlled trial comparing surgery to nonoperative treatment. However, most of the surgery performed involved accepted interventions . At baseindex scores between the responders and the non-responders. However, the responders tended to be women and to be older, causing our results to be a conservative interpretation. In this analysis, no information on patient co-morbidities or on other types of interfering conditions was collected. Thus, the study can be considered to represent a cross-section of orthopedic patients who undergo surgery at a university hospital.The department only mailed 1 follow-up questionnaire to the patients and no reminders, which led to a loss of more than 40% of those initially included in the study. In the dropout analysis, no major difference was found in the preoperative EQ-5D We selected 1 year as a time outcome measurement because it was an easy endpoint. In some groups of patients (e.g. elderly), it might have been better to have had a shorter time frame because many other factors may have impaired the results.The choice of algorithm used to convert self-classification scores can affect the index-based score, as shown in a study that compared UK and US scoring algorithms in patients undergoing percutaneous coronary intervention (PCI) . HoweverThe EQ-5D instrument has potential limitations. It may lack responsiveness to small but clinically important changes in health . In the The bimodal distribution of EQ-5D scores that we found preoperatively and at the 12-month follow-up has also been reported by others . The EQ-We consider that our cohort represents patients in general who have undergone orthopedic surgery. This is the largest orthopedic cohort to be studied regarding HRQL so far, with 426 diagnoses and 446 orthopedic procedures. It could be questioned why we divided the cohort into 15 groups according to anatomical region and type of surgery, but it would have been difficult to present the results in any other way due to the large number of procedures. The drawback of this is that we lost the possibility of presenting details of specific diagnoses and procedures. We notice that our large cohort had a low HRQOL according to EQ-5D index score. A major strength in our report is that we matched our cohort with the Swedish EQ-5D reference population survey , 2003. W